This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ABC transporters are one of the largest and most diverse super-families of membrane proteins found in all living organisms ranging from bacteria to humans.1-5 These membrane proteins act as mechanical pumps that couple ATP hydrolysis to either uptake or export of a wide diversity of substrates, including ions, sugars, lipids, peptides or complex organic molecules, against concentration gradients, across biological membranes.1-5 Interestingly, all ABC transporters share a common organization of a four core domains: two transmembrane domains (TMD) and two nucleotide-binding domains (NBD), indicating a common evolutionary origin and suggesting a similar mechanism of energy coupling. ABC transporters are medically relevant because they can be responsible for antibiotic and antifungal resistances in micro-organisms, resistance to chemotherapeutic treatments in cancer cells, and dysfunctions of many eukaryotic members are the molecular basis for severe sicknesses (e.g., cystic fibrosis).5-7 Unfortunately, despite extensive studies, many questions about the molecular mechanism and interactions of ABC transporters remain unanswered. Built upon the significant progress that we have made and the recent development of cryo-transmission electron microscopy (cryo-TEM), in this revised proposal, we aim to expand our original proposed research scope to use cryo-TEM, computer reconstruction and structure mining to solve the structure and molecular mechanism of BmrA, a multidrug bacterial transporter belonging to the ABC transporter superfamily, by collaborating with the National Center for Macromolecular Imaging (NCMI) (a NCRR designated Biomedical Research Resource for Structural Biology, at http://ncmi.bcm.tmc.edu/).